The leading cause of death associated with cancer is tumor metastasis. Metastasis is the ability of malignant cells to leave the primary tumor, migrate to distant sites in the body and establish secondary tumors. From a clinical standpoint, the prevention or inhibition of metastasis is vital to the treatment of cancer. S100A4, a member of the S100 family of calcium-binding proteins, regulates carcinoma cell motility via interactions with myosin-II. Numerous studies indicate that S100A4 is not simply a marker for metastatic disease, but rather has a direct role in metastatic progression. These observations suggest that S100A4 is an excellent target for therapeutic intervention. We used a novel biosensor-based assay to identify a series of small molecule inhibitors of S100A4 and defined the atomic determinants responsible for binding by high resolution x-ray crystallography. These studies are supporting the development of second generation S100A4 inhibitors with enhanced affinity and selectivity. We will complement our biosensor-based assay with a fluorescence polarization assay to identify small molecule inhibitors that directly disrupt the S100A4/myosin-IIA interaction. Lead compounds from both assays will be characterized and optimized. Structural studies will identify the chemical and structural determinants involved in S100A4 inhibition. Biochemical analyses will examine the selectivity and potency of lead compounds as well as the mechanisms by which small molecule inhibitors prevent S100A4 activation and interfere with S100A4-mediated regulation of myosin-IIA assembly. Cell-based studies will provide proof-of-principle that S100A4 inhibitors affect the motile and invasive capabilities of carcinoma cells. Using animal models of breast cancer, we will examine the efficacy of S100A4 inhibitors to limit and/or inhibit metastasis. The outcome of these studies will be the identification and characterization of compounds that may serve as therapeutic leads for the treatment of metastatic cancer. PUBLIC HEALTH RELEVANCE: The leading cause of death associated with cancer is tumor metastasis. From a clinical standpoint, the prevention or inhibition of metastasis is vital to the treatment of cancer. Numerous studies indicate that S100A4, a member of the S100 family of calcium-binding proteins, has a direct role in metastatic progression. These observations suggest that S100A4 is an excellent target for therapeutic intervention. Our proposed studies address the development and testing of potent and selective S100A4 inhibitors. The completion of these studies will be the identification and characterization of compounds that may serve as therapeutic leads for the treatment of metastatic cancer. [unreadable] [unreadable] [unreadable]